Precise control of protein synthesis is essential for maintenance of normal cellular function and is central to innate antiviral responses within the cell. For example, the innate immune system protein 2'-5'-oligoadenylate synthetase (OAS) detects cytosolic double-stranded (ds)RNA to initiate a translational control response, via activation of the latent ribonuclease L (RNase L), which limits viral protein synthesis and thus replication. Structures of OAS1 and OAS1-dsRNA complexes have revealed important insights into OAS1 activation: dsRNA binding drives a functionally essential reorganization of the OAS1 active site. However, our recent discovery of a novel single-stranded RNA motif which strongly potentiates activation of OAS1, and extensive preliminary data presented here, strongly argue that we still have limited understanding of how specific RNA features and their contexts combine to drive potent activation of OAS1. Our new data show that the model dsRNA used for OAS1 structural studies contains competing activating and non-activating OAS1 binding sites, and that currently ill-defined RNA feature(s) direct binding orientation in solution and thus control the potency of OAS1 activation. Further, we show that the human non-coding RNA 886 (nc886) contains a novel RNA tertiary structure that is a unique and remarkably potent activator of OAS1. This proposal describes an innovative, multidisciplinary study with a specific focus on defining the RNA features and contexts responsible for driving OAS1 activation and their resultant impacts on the cellular antiviral response. In Aim 1, we will use sequence and length variants of a model dsRNA to decipher the ?rules? that govern OAS1 activation by dsRNA. Using in vitro biochemical and human cell-based assays coupled with biophysical, proteomic, and structural approaches, we will determine how specific RNA signatures work, cooperatively or in competition, to drive OAS1-dsRNA interaction and the extent of OAS1 activity. Complementary virological assays will place this new understanding of dsRNA-mediated regulation of the OAS/RNase L pathway, and thus resistance to viral infection, in an appropriate biological context. In Aim 2, we will determine the molecular feature(s) of nc886 that lead to its potent activation of OAS1. Further, we will test our novel hypothesis that upregulation of nc886 during influenza A infection is specifically countered by interaction with the viral NS1 protein. Collectively, these studies will reveal novel insights into RNA-mediated translational control via the OAS/RNase L pathway that may serve as a framework to define the biological role(s) of natural OAS1 activators such as nc886 and the OAS1 evasion strategies adopted by diverse viruses. Such knowledge will be an essential foundation for development of generally applicable anti-viral therapeutic approaches and can inform strategies for treatment of other human diseases, for example by activating the OAS/RNase L pathway as a novel route to control the proliferation/ adhesion characteristic of metastasis.